The dental pulp is a complex and highly vascularized tissue that houses specialized cells (i.e. odontoblasts) and undifferentiated cells named dental pulp stem cells (DPSC). DPSC constitute a subpopulation of cells endowed with multipotency and self-renewing capacity that are vital in the homeostasis and regeneration of the dental pulp. While numerous factors and pathways regulate stem cell fate, the mechanisms that maintain cells in undifferentiated states remain unclear. Bmi-1, a component of the polycomb repressive complex 1 (PRC1) is expressed in high levels in self-renewing stem cells and inhibits cell cycle regulators (p16Ink4a and p19Arf). Recent studies demonstrate that DPSC are found primarily in the perivascular niche (Shi et al., 2003). Our preliminary results indicate that conditioned media from endothelial cells induce and sustain the expression of Bmi-1 in DPSC, and that recombinant human Platelet-Derived Growth Factor-BB (PDGF-BB) induces Bmi-1 in a dose-dependent manner. Here, we hypothesize that endothelial cell-derived factors induce Bmi-1 and enhance self-renewal of DPSC. To address this hypothesis, we propose the following specific aims: S.A.#1) to determine the role of endothelial cell-derived factors in Bmi-1 regulation in DPSC. PDGF-BB is a potent mitogen secreted by endothelial cells. Characterization of transgenic mice of PDGFRa, a co-receptor for PDGF-BB, shows abnormal tooth development, suggesting a role for this signaling pathway in the determination of stem cell fate (Xu et al., 2005). Thus, we hypothesize that endothelial cell-derived PDGF-BB regulates Bmi-1 signaling in DPSC. S.A.#2) to define the functional role of Bmi-1 on the self-renewal of DPSC. Preliminary data utilizing a well-established in vitro model of self-renewal showed the ability of DPSC to form spheroid bodies in ultra-low attachment conditions, suggesting self-renewal capacity. We hypothesize that Bmi-1 is functionally necessary for DPSC self-renewal. S.A.#3) to determine the impact of Bmi-1 function in DPSC on dentinogenesis in engineered dental pulps. It has been shown that Bmi-1 knockout mice present thin dentinal walls, indicating a functional role for Bmi-1 in dentin formation (Biehs et al., 2013). Here, we hypothesize that Bmi-1 is necessary for DPSC-mediated dentin regeneration. In summary, the studies proposed here will define the role of Bmi-1 on the self-renewal and regenerative capacity of dental pulp stem cells. Successful completion of these studies will define key mechanisms underlying the maintenance of a stem cell pool in dental pulps, and may unveil a novel pathway that can be engaged therapeutically in Regenerative Endodontics.